Synaptic transmission and its regulation are critical processes for nervous system function, including learning and memory, and abnormal neurotransmission can cause neurological disorders such as epilepsy and impaired cognitive abilities. Arrival of an action potential at a presynaptic site causes a spike in calcium levels that induces synaptic vesicle release which is synchronous with the action potential. The molecular sensors that read Ca level changes and initiate this release are synaptotagmins 1, 2 and 9 which bind Ca through two C2 domains. During high-frequency neuronal firing, resting Ca levels rise significantly as the Ca extrusion mechanisms are overwhelmed and a second mode of neurotransmission release is observed, one that is asynchronous and does not depend on synaptotagmins 1, 2 and 9. Elevated resting Ca levels can also mediate changes in synaptic plasticity. Two candidate Ca sensors to regulate these two processes are the Doc2 proteins since, like synaptotagmins, they contain two C2 domains and associate with lipids in a Ca-dependent manner. Indeed, preliminary evidence supports a Doc2 role in neurotransmission. This proposal seeks to employ shRNA to perform knockdown of Doc2A and Doc2B in mouse cortical cultures and to assay synaptic function in these cultures. Specifically, the second aim will asses basal transmission in shRNA-treated cultures by measuring evoked inhibitory and excitatory postsynaptic potentials (IPSCs and EPSCs) and spontaneous miniature ISPCs. The main goal will be to examine if absence of Doc2 results in (1) altered short-term synaptic plasticity as determined by changes in the paired-pulse ratio or short-term depression during a 10 Hz stimulus, and in (2) reduction of asynchronous release in cultures derived from synaptotagmin 1 null mouse which lack most synchronous release. To asses which Doc2 C2 domains are important for function, biochemical assays will be performed to measure the Ca-dependent phospholipid binding affinity of each domain. As a last aim, any synaptic defects observed will be rescued with wild-type Doc2 or mutant constructs that cannot bind Ca. These experiments should yield insight into basic features of neurotransmission and how its dysfunction might lead to neurological defects. Communication between neurons in our brains relies heavily on regulation of calcium concentrations. Abnormal neuronal communication can result in neurological conditions such as epilepsy. Insight into how this communication is affected in neurological diseases might be gained by characterizing Doc2 proteins which may act as calcium sensors to regulate neuronal communication.